Browsing by Subject "Retina"
Now showing 1 - 13 of 13
Results Per Page
Sort Options
Item Antigen presenting cells In the retina(2010-10) Lehmann, UteThe presence and activity of dendritic cells (DC) in retina is controversial. This is in part due to the limited number of DC in the retina and the small number of reliable DC markers. In addition, functions ascribed to DC in other parts of the body are thought to be done by microglia in the central nervous system. CD11c is the cellular marker most associated with DC. In order to study the nature of retinal DC, transgenic mice that express green fluorescent protein (GFP) on the CD11c promoter were used to visualize, quantify, and characterize DC in both quiescent and injured retinas. In the quiescent retina it was found that compared to the number of neurons and microglial cells there were relatively few DC located in the nerve fiber layer and the outer plexiform layer. Upon retinal injury by either optic nerve crush or exposure to continuous bright light, the numbers of DC increased significantly and translocated to retinal layers associated with the injury. Surprisingly, the retinas of the eyes contralateral to the optic nerve crush also showed a significant increase in DC. The potential origin of the DC in retina was examined using a chimeric mouse paradigm. Most of the retinal DC were found to originate from circulating precursors, and a smaller number from a local progenitor cell population. This study suggests that retinal DC are a previously overlooked population, distinct from microglia, and may be important in the injury response and maintenance of homeostasis in the retina.Item Characterization of blood flow in the retinal vascular network(2015-01) Kornfield, Tess EllenThe primary goal of the work presented here is to understand how blood flow is regulated in the retinal vascular network in response to neuronal activity. In order to accurately quantify blood flow, we developed a multitude of streamlined techniques capable of measuring many properties of blood flow. These techniques were used to investigate retinal functional hyperemia, defined as the increase in local blood flow that occurs in response to nearby neuronal activity. We did a comprehensive survey of all retinal vessels to investigate the magnitude and timing of the functional hyperemia response as it presents in the different compartments of the retinal vascular network. We found that arterioles are primarily responsible for generating functional hyperemia in the retina and that, with prolonged stimulation, blood flow through the three vascular layers in the retina is differentially regulated. This result implies the presence of active capillary dilation. The work in this dissertation informs our understanding of blood flow regulation within the retinal vascular network.Item D-serine: a study of its function and regulation in the retina.(2009-10) Gustafson, Eric CharlesActivation of the NMDA-type glutamate receptor requires the simultaneous binding of both glutamate and a coagonist, either glycine or D-serine. In the inner retina, glutamate released from bipolar cells excites NMDA receptors on retinal ganglion cells and some amacrine cells. The identity of the coagonist, however, has remained unknown. Early on, the relatively high levels of glycine and its use in the retina as an inhibitory transmitter by a subset of amacrine cells led many to believe that glycine was the endogenous coagonist. The discovery that D-serine and its synthesizing enzyme, serine racemase, are both present in the retina suggested that D-serine may play a role as well. This manuscript reports results that have examined the role of D-serine in the retinas of larval tiger salamanders and in mice. These studies suggest that D-serine is the major endogenous coagonist during light-induced responses in the inner retina of both species. In addition, the regulatory mechanisms of glycine transport and of the endogenous D-serine degrading enzyme, D-amino acid oxidase, have been shown to be essential in maintaining coagonist levels below that needed to saturate the NMDA receptor. Together, the results position D-serine as a major contributor and potential modulator of excitatory neurotransmission in the retina.Item Dynamic regulation of the NMDA receptor coagonist D-serine in the mammalian retina.(2011-09) Sullivan, Steven J.The N-methyl D-aspartate (NMDA) receptor coagonist D-serine is important in a number of different processes in the central nervous system, ranging from synaptic plasticity to disease states, including schizophrenia. In the retina, light-evoked responses of retinal ganglion cells are shaped in part by NMDA receptors which require a coagonist for activation. There is debate over whether glycine or D-serine is the endogenous coagonist of retinal ganglion NMDA receptors. I used a mutant mouse lacking functional serine racemase (SRKO), the only known D-serine synthesizing enzyme in mammals, to show that retinal ganglion cells depend on D-serine for NMDAR activation (chapter 1). Most changes in NMDA receptor currents during synaptic activity have been attributed to glutamate fluctuations against a steady background of coagonist, excluding the possibility of dynamic coagonist release. The retina is a particularly useful system to determine if coagonist release occurs in the nervous system, because it can be naturally stimulated with light. By saturating the glutamate binding site of NMDA receptors, I was able to measure coagonist release during light-evoked responses. Coagonist release was detected in retinal ganglion cell light responses and depended on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic AMPA receptors. Coagonist release was significantly lower in SRKO mice (chapter 2). By directly measuring extracellular D-serine using capillary electrophoresis, I demonstrated that D-serine can be released from the intact mouse retina through an AMPA receptor dependent mechanism (chapter 3). The collective works put forth in this thesis imply that activity-dependent modulation of D-serine availability may add an extra dimension to NMDA receptor coincidence detection in the central nervous system.Item Identifying novel roles for the immunoproteasome in the retina.(2010-10) Hussong, Stacy AnnImmunoproteasome is a proteasome sub-type that is known to produce antigenic peptides for MHC class I presentation. However, immunoproteasome is present in the immune-privileged brain and retina and is upregulated with disease in human retina and injury in mouse retina and brain, suggesting functions unrelated to its role in the immune system. The goal of this thesis is to define novel roles for the immunoproteasome in the retina. Potential functions of the immunoproteasome were defined by comparing the stress response of wild-type and knock-out mice missing one (lmp7-/-(L7)) or two (lmp7-/- /mecl-1-/-(L7M1)) of the three immunoproteasome subunits. Aging was used as a model system for chronic stress. Chronic peroxide exposure in cultured retinal pigment epithelial (RPE) cells developed from wild-type mice was used as an additional stress model. In wild-type retinas and RPE cells, upregulation of immunoproteasome was observed in response to both models of chronic stress. To determine the consequence of eliminating immunoproteasome, the retinas and RPE cells from KO mice were examined.L7M1 retina had significantly elevated levels of photoreceptor apoptosis that further increased with age. In addition, L7M1 cell lines were more susceptible to oxidantinduced death. Together these data suggest immunoproteasome is protective against oxidative stress. The localization of immunoproteasome to the outer plexiform layer in wild-type retina suggested a role in retinal function. Electroretinography was used to test the hypothesis that immunoproteasome is required for maintaining normal visual transmission. Data indicated that immunoproteasome-deficient mice had a decreased bipolar cell response as compared to wild-type. Evaluation of several retinal synapse proteins by Western blot revealed no significant difference in protein content across strains. In addition, gross retinal morphology and bipolar cell density were not different. In conclusion, immunoproteasome-deficiency causes a decrease in visual transmission but the mechanism is still unclear. In summary, these data provide compelling evidence that immunoproteasome has a role in retinal stress response, specifically in protecting against oxidative stress. Furthermore, immunoproteasome-deficient mice have a decreased bipolar cell response as measured by ERG. Altogether, data from this thesis strongly support the hypothesis that immunoproteasome has additional functions in the retina that do not involve immune function.Item Intrinsically photosensitive retinal ganglion cells: diversity of form and function.(2010-12) Schmidt, Tiffany M.A subpopulation of retinal ganglion cells (RGCs) express the photopigment melanopsin, rendering them intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors are critical for several non-image forming behaviors including circadian entrainment and the pupillary light reflex. Initially thought to be a uniform population, later studies demonstrated that there was at least some degree of morphological and physiological diversity in the ipRGC population. Technical limitations, however, had prevented the comprehensive study of ipRGCs at the single cell level. The purpose of this project was to utilize a mouse model in which ipRGCs are labeled in vivo with enhanced green fluorescent protein to identify and target single ipRGCs for morphological and physiological analyses. The central hypothesis of the research presented herein is that distinct morphological ipRGC subtypes have distinct physiological properties and synaptic inputs, resulting in unique light information sent to target nuclei in the brain by the various ipRGC subpopulations. This work has confirmed the existence and further analyzed the morphological and physiological properties of at least three ipRGC subtypes: M1 cells with dendrites stratifying in the OFF sublamina of the inner plexiform layer (IPL), M2 cells with dendrites stratifying in the ON sublamina of the IPL, and M3 cells with dendrites bistratifying in both the ON and OFF sublaminas of the IPL. We find that these cell types do indeed possess distinct intrinsic light responses and intrinsic membrane properties. Furthermore, we find that these subpopulations are differentially influenced by cone-mediated signals. Finally, we find that the cation channel involved in ipRGC signal transduction is not composed solely of the canonical transient receptor potential channel (TRPC) subunit 3, 6, or 7. However, we do find that TRPC6 is involved in mediating the melanopsin-evoked light response in both M1 and M2 cells, with both subtypes showing a reduction in the magnitude of the intrinsic light response in TRPC6-/- animals. Collectively, the differential influence of intrinsic, melanopsin-mediated phototransduction and synaptically-evoked extrinsic inputs on the integrated light-evoked response of ipRGC subtypes indicates that these subtypes may serve as conduits for distinct light information sent to the brain. We discuss the implications of these findings and propose a model for the differential influence of distinct ipRGC subtypes on various non-image forming behaviors.Item Investigation of the intranasal delivery method as a means of targeting therapeutic agents to the Injured retina and optic nerve.(2009-09) Alcalá, Sandra R.Ischemic optic neuropathy (ION) is a visually devastating disease process in which there is disruption of arterial blood flow to the optic nerve head. ION, both anterior and posterior, is the most common cause of sudden optic nerve-related vision loss in the developed world. In addition, traumatic optic neuropathy (TON), caused by blunt trauma to the orbit and/or face, can cause tractional, compressive, or ischemic injuries to the optic nerve as well. These types of injuries to the optic nerve can ultimately result in the death of retinal ganglion cells in the retina and, consequently, the functional loss of vision. Currently, there is no effective treatment for these types of injury. There are several issues that stand in the way of adopting treatment modalities for injuries to the optic nerve and retina. Many potential therapeutic drugs are unable to gain access to the affected cells due to the protective blood-retinal and blood-brain barrier. Neurotrophic factors are endogenous large molecular weight neuroprotective proteins that, upon injury, are released in an autocrine and paracrine fashion to reduce apoptotic cellular death. However, these factors possess inherent molecular characteristics that impede their transport through the protective blood-brain barrier. Therefore, the ability to bypass the blood-brain barrier using a non-invasive means would have great clinical potential. This study examined the viability of the intranasal delivery method as a means of targeting therapeutic agents to the injured retina and optic nerve.Item Mechanisms and functional consequences of glial signaling in the retina.(2009-07) Kurth-Nelson, Zebulun LloydTwenty years ago, glia were viewed as passive support cells for neurons. Since then, experiments have shown that glial cells have their own form of excitability with precise intracellular spatiotemporal dynamics, intercellular communication among themselves, a bidirectional dialog with neurons and synapses, and a key role in mediating blood flow changes in response to neuronal activity. Most of these experiments have been conducted in brain regions such as hippocampus, cortex, hypothalamus, and cerebellum. However, as work from our laboratory has shown, the mammalian retina is also an excellent preparation to study the active functions of glial cells. Here, we describe two forms of active glial signaling in the retina. First, we tested the hypothesis that glial cells modulate synaptic activity in the retina. We measured synaptic strength by evoking excitatory postsynaptic currents (EPSCs) in ganglion cells with either light or an electrical stimulus. We then excited glial cells through several methods, including agonist ejection, photolysis of caged Ca2+, and depolarization. The amplitude of the synaptic currents was altered by some, but not all, of these glial stimuli, leaving us unable to draw a definitive conclusion as to whether glial excitation alone is sufficient to modulate synaptic transmission in the retina. Second, we characterized spontaneous intercellular glial Ca2+ waves in the retina. Glial cell excitability takes the form of transient intracellular Ca2+ elevations. One of the first recognized active properties of glia was their ability to propagate these Ca2+ elevations from cell to cell in a wave-like pattern. In most previous experiments, glial Ca2+ waves were initiated by an experimenter-driven stimulus, raising doubts about whether these waves occurred naturally in the organism. We demonstrate here that these waves occur spontaneously both in intact tissue and in vivo, and that the rate of spontaneous wave generation increases as animals age. These spontaneous waves propagate by glial release of ATP and activation of ATP receptors on neighboring cells. Finally, spontaneous waves cause changes in blood vessel diameter. This is the first demonstration of a functional effect of spontaneous intercellular glial signaling. These results suggest a functional role for glial cell signaling in the retina and raise the possibility that glial signaling may actively participate in the aging of the nervous system.Item Mechanisms of Blood Flow Regulation in the Retina: Glial Calcium Signaling Regulates Capillary, but Not Arteriole Diameter(2016-12) Biesecker, KyleBlood flow is tightly regulated in the central nervous system to ensure neurons receive sufficient oxygen and glucose. When neuronal activity increases, nearby blood vessels dilate to increase local blood flow, a phenomenon termed functional hyperemia. Two key controversies have arisen concerning the mechanisms that underlie functional hyperemia. Firstly, the role of glial Ca2+ signaling in triggering vessel dilations is unclear. Some evidence suggests that glial Ca2+ signals precede vessel dilations, but blocking glial Ca2+ signaling does not alter functional hyperemia. Secondly, data has been presented arguing both for and against the ability of capillaries to actively dilate during functional hyperemia. Herein, I demonstrate that glial Ca2+ signaling does play a key role in regulating capillary diameter, but is not necessary for regulating arteriole diameter. Additionally, capillaries can actively dilate during functional hyperemia responses. These findings suggest that glial Ca2+ signaling contributes to blood flow regulation in the central nervous system by triggering capillary dilations during functional hyperemia.Item Modulation of neurovascular coupling in the retina:effects of oxygen and diabetic retinopathy.(2011-07) Mishra, AnushaNeurovascular coupling is a process by which neuronal activity leads to localized increases in blood flow in the central nervous system. When neurovascular coupling results in hyperperfusion of the neural tissue, the response is termed functional hyperemia and serves to satisfy the increased energy demand of active neurons. In brain slices, high [O2] alters neurovascular coupling, decreasing activity-dependent vasodilations and increasing vasoconstrictions. However, in vivo, hyperoxia has no effect on neurovascular coupling. In order to resolve these conflicting reports of O2 modulation, I examined neurovascular coupling in both ex vivo and in vivo rat retina preparations. In the ex vivo retina, 100% O2 reduced the amplitude of light-evoked arteriole vasodilations by 3.9-fold and increased the amplitude of vasoconstrictions by 2.6-fold when compared to responses in atmospheric [O2] (21%), consistent with slice data. Oxygen exerted its effect by decreasing vasodilatory prostaglandin signaling and increasing vasoconstrictory 20-hydroxyeicosatetraenoic acid signaling. However, in vivo, hyperoxia (breathing 100% O2) had no effect on light-evoked arteriole vasodilations or on blood flow. We found that the differing effects of O2 arise because retinal pO2 increases to a much greater extent in the ex vivo preparation (to 548 mmHg) than in vivo (to 53 mmHg; Yu et al. Am J Physiol 267:H2498-H2507). When retinal pO2 was raised to 53 mmHg in the ex vivo retina, no change in neurovascular coupling was observed. These results demonstrate that although O2 can modulate neurovascular signaling pathways when pO2 is raised high enough, such levels are not attained in vivo, even when an animal breaths 100% O2. Functional hyperemia can also be modulated by pathological conditions. It is diminished in the retinas of diabetic patients, possibly contributing to the development of diabetic retinopathy. I investigated the mechanism responsible for this loss in a streptozotocin-induced rat model of type 1 diabetes. Here I show that light-evoked arteriole dilation was reduced by 58% in these diabetic rats at 7 month survival time. The diabetic retinas showed neither a decrease in the thickness of the retinal layers nor an increase in neuronal loss, although signs of early glial reactivity were observed. Functional hyperemia is believed to be mediated, at least in part, by glial cells and we found that glial-evoked vasodilation was reduced by 60% in diabetic animals. An upregulation of inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry, and inhibition of iNOS restored both light- and glial-evoked dilations to control levels. These findings suggest that high NO levels resulting from iNOS upregulation alters glial control of vessel diameter and may underlie the loss of functional hyperemia observed in diabetic retinopathy. I further tested whether inhibiting iNOS reverses the loss of flicker-induced vasodilation in diabetic rat retinas in vivo. Flicker-evoked arteriolar dilations were diminished by 61% in diabetic animals, compared to non-diabetic controls. Treating diabetic animals with aminoguanidine (an iNOS inhibitor), either acutely via IV injection or long-term in drinking water, restored flicker-induced arteriole dilations in diabetic rats to control levels. The amplitude of the electroretinogram b-wave was similar in control and diabetic animals, suggesting that the deficit in functional hyperemia was not due to a reduction in neuronal activity. These findings demonstrate that inhibiting iNOS with AG is effective in preventing the loss of, and restoring, normal flicker-induced vasodilation in the diabetic rat retina. Treatment with iNOS inhibitors early in the course of diabetes has the potential to slow the progression of retinopathy by maintaining normal neurovascular coupling.Item Modulation of the Junctional Conductance of Retinal AII Amacrine Cell Electrical Synapses(2023-10) Cable, ChloeRetinal AII amacrine cells are extensively coupled together by electrical synapses. Changes to the strength of these synapses affect how signals are routed through rod and cone retinal pathways during scotopic and photopic vision. Plasticity at these electrical synapses have not, to date, been characterized using electrophysiological approaches. We investigated the effects of adenosine (AR) and N-methyl-D-aspartate receptor (NMDAR) activation on the electrical coupling between AII cells using dual whole-cell patch-clamp electrophysiology in mouse retinal slices. While neither AR activation nor inhibition affected junctional conductance, NMDAR activation substantially decreased junctional conductance between AII cells. Relieving the Mg2+ block of NMDARs through bath application of Mg2+-free solution or by depolarizing AII cells to 0 mV reduced junctional conductance. Exogenous application of NMDA decreased conductance between cells, a decrease which was blocked by the non-selective NMDAR antagonist APV but not by Ro 25-6981, a selective GluN2B-NMDAR antagonist. Addition of either D-serine or glycine, both NMDAR coagonists, without NMDA, reduced the junctional conductance and addition of either coagonist to NMDA-treated retinas further decreased conductance. Experiments were conducted in inositol 1,4,5-trisphosphate receptor type 2 KO and serine racemase KO mice and in WT mice with D-amino acid oxidase to reduce retinal D-serine levels. Under these conditions, the NMDA-mediated conductance decrease was maintained, indicating that D-serine is not necessary for NMDAR-mediated plasticity. These results demonstrate that NMDAR activation results in a decrease in electrical coupling between AII amacrine cells and suggests that both D-serine and glycine can serve as NMDAR coagonists for this plasticity.Item Novel Biomarker Identification Approaches for Schizophrenia using fMRI and Retinal Electrophysiology(2017-11) Moghimi, PanteaSchizophrenia is a chronic mental illness. The exact cause if schizophrenia is not yet known. Extensive research has been done to identify robust biomarkers for the disease using non-invasive brain imaging techniques. A robust biomarker can be informative about pathophysiology of the disease and can guide clinicians into developing more effective interventions. The aim of this dissertation is two folds. First, we seek to identify robust biomarkers using resting state fMRI activity from a cohort of schizophrenic and healthy subjects in a purely data driven approach. We will calculate multivariate network measures and use them as features for classification of the subjects into healthy and diseased. The network measures will be calculated using nodes defined by the AAL anatomical atlas as well as a functional atlas constructed from the fMRI activity. Network measures with high classification rate may be used as potential biomarkers. We will employ double cross-validation to estimate generalizability of our results to a new population of subjects that were not used in biomarker identification. Second, we seek to identify biomarkers using electroretinogram (ERG). We will use a data driven approach to classify individuals based on the pattern of retinal activity they exhibit in response to visual stimulation. Characteristics of the ERG result in high classification rate are presented as potential biomarkers of schizophrenia.Item Progenitor cell maturation and initiation of neurogenesis in the developing vertebrate neural retina.(2009-10) Yang, Hyun-JinThe mature vertebrate central nervous system is composed of an enormous number of neuronal and glial cells. A relatively small number of progenitor cells generate these cells during a finite period of time of development. Progenitor cells during early stages of central nervous system development divide so that each division produces two progeny that divide again. This `preneurogenic' mode of division is essential for the exponential increase of number of progenitor cells. Later, progenitor cells change their mode of division to `neurogenic', where one or both daughter cells produced by a division withdraw from the mitotic cycle and differentiate. This more mature, neurogenic division is critical for generation of a functional nervous system. The aim of the project described in this thesis was to understand: 1) the molecular differences that dictate the two modes of progenitor cell division, namely preneurogenic and neurogenic, 2) the mechanism that regulates the switch in the mode of division, and 3) the molecular trigger that initiates differentiation. Molecular differences between preneurogenic and neurogenic progenitor cells were identified, and are described in more detail in chapter II. The early, preneurogenic progenitor cells express the transcription factor, Sox2, and a ligand for the Notch receptor, Delta1. The more mature, neurogenic progenitor cells express Sox2 and the bHLH transcription factor, E2A, and do not express Delta1. Perturbation of Notch signaling resulted in conversion of progenitor cells from preneurogenic to neurogenic and in premature neurogenesis. Furthermore, Sonic hedgehog was found to be expressed by a subset of newly differentiating cells. Misexpression of Sonic hedgehog led to premature maturation of preneurogenic progenitor cells and neurogenesis. These results suggest that Notch signaling maintains progenitor cells in the preneurogenic state and that Sonic hedgehog initiates progenitor cell maturation. Certain proneural bHLH transcription factors were found to initiate neurogenesis, and are described in more detail in chapter III. Expression of a number of proneural bHLH factors comes up in a stereotypic temporal sequence prior to the onset of ganglion cell differentiation. Ascl1 and Neurog2 were expressed first, which was followed by expression of Neurod1 and Neurod4. Finally, Atoh7 was expressed, which preceded the appearance of ganglion cells. Individual progenitor cells expressed heterogeneous combinations of proneural genes prior to ganglion cell genesis. Misexpression of Ascl1 or Neurog2 in preneurogenic retina was sufficient to initiate ganglion cell genesis. Misexpression of Neurog2 initiated the stereotypic sequence of proneural gene expression that normally preceded ganglion cell genesis. Ascl1 expression was also sufficient to initiate ganglion cell genesis. However, it functioned by a mechanism distinct from that of Neurog2. These results suggest that ganglion cell genesis may be initiated by two different mechanisms.